Toxin and antitoxin (TA) systems are commonly found in prokaryotes. These systems function to allow the organisms to rapidly adjust rates of protein and DNA synthesis in order to respond to external stimuli and/or stress (Gerdes et al. 2005. Nature Rev. Microbiol. 3:371-382). Under normal circumstances, TA genes are co-transcribed and co-translated as part of an operon so that both antitoxin and toxin are produced together within the cytosol to form an inert complex. Under specific stress, transcription of the TA promoter will be repressed, disrupting transcription and subsequently translation. As toxins are stable compounds while the antitoxins compounds are more labile and prone to proteolytic attack by bacterial Lon/ClpP proteases, disruption of transcription from the TA promoter results in excess toxin and activity of the toxin in the cell. The target of such toxins may be mRNA, DNA gyrase or DNA helicase, where interaction of toxin with these targets leads to disruption of transcription and translation of genes responsible for important cellular processes.
Based on sequence homology and cellular targets, there are eight major TA systems that have been identified in prokaryotes (Gerdes et al. 2005. Nature Rev. Microbiol. 3:371-382; Kamphius et al. 2007. Protein Peptide Lett. 14:113-124). Among these TA systems is the MazEF system, which includes the toxin MazF and the antitoxin MazE. These two proteins form a linear heterohexamer made up of alternating toxin and antitoxin homodimers (Kamada et al. 2003. Mol. Cell. 11:875-884). The MazEF TA complex in E. coli has been shown to autoregulate by binding of the DNA by the N-terminal domain of MazE. The MazF toxin has been shown to cleave translated mRNAs and through this mechanism to block protein synthesis within prokaryotic cells (Christensen et al. 2003. J. Mol. Biol. 332:809-819). The cleavage of mRNAs in E. coli is at ACA sites (Zhang et al. 2003. Mol. Cell. 12:913-923). A variety of conditions have been shown to trigger the activity of MazF in prokaryotic cells including for example, stress linked to high temperatures, oxidative stress, DNA damage by thymidine starvation, UV irradiation, and contact with protein-inhibiting antibiotics (Kamphius et al. 2007. Protein Peptide Lett. 14:113-124). Although MazEF clearly functions as a bacteriostatic system (Gerdes et al. 2005. Nature Rev. Micrabiol. 3:371-382) within prokaryotic cells, it is not clear whether MazEF also functions within cells as a system for programmed cell death.
Sequence analysis has revealed that the MazF toxin is more conserved among different bacteria than is the antitoxin MazE. This finding is consistent with the finding that the activity of similar TA systems in different bacteria is dependent on the specificity of the antitoxin. In fact, it has been found that Staphylococcus aureus MazEF homologs are quite different from E. coli MazEF homologs (Fu et al. 2007. J. Bacteriol. 189:8871-8879; Fu et al. 2009. J. Bacteriol. 191:2051-2059; Niles et al. 2009. J. Bacteriol. 191:2795-2805). It has been found through transcriptional analysis that the mRNA target of the toxin MazF in S. aureus is selective, sparing important transcripts such as gyrA and recA (Niles et al. 2009. J. Bacteriol. 191:2795-2805). Therefore, in S. aureus MazF has features of a bacteriostatic effect rather than a bacteriocidal effect. The effect can be reversed by MazE, but only within a specific time window beyond which the cells would become nonviable.
Based on the importance of TA systems within cells, including the MazEF system, interest has grown in the use of these systems in the development of new antibiotic compounds. To date, the only organisms not identified as having MazEF systems are Mycobacerium leprae, Chlymidia, Rickettsia, and Mycoplasm. MazEF has been found to be an important TA system within a variety of prokaryotes including E. coli, S. aureus, and S. pneumonia. There remains a need for new antibiotic compounds active against clinically important bacteria.